Thiazine dyes used to inactivate HIV in biological fluids

ABSTRACT

A method for using thiazine dyes, especially methylene blue, alone or in combination with low levels of light, to selectively inactivate or inhibit intracellular replication of specific viruses, especially human immunodeficiency virus in vitro and in vivo. Examples of useful thiazine dyes are methylene blue, azure A, azure C, toluidine blue O, and thionine. The preferred dye at this time is methylene blue. Methylene blue is FDA approved for topical, i.v., and oral administration, and has minimal side effects. Since methylene blue absorbs in the red wavelengths, i.e., approximately 670 nm, which penetrates tissue much better than other lower wavelengths, light penetrating the skin to the capillaries at the surface can be used to enhance the activity of the dye.

The United States government has rights in this invention by virtue ofNational Institutes of Health grant No. CA42854.

BACKGROUND OF THE INVENTION

This is a continuation of U.S. Ser. No. 07/632,606, filed Dec. 24, 1990,now abandoned which is a continuation of U.S. Ser. No. 07/350,383, filedMay 11, 1989 now abandoned which is continuation-in-part of U.S. Ser.No. 264,088 entitled "Phototherapy Using Methylene Blue" filed Oct. 28,1988, now U.S. Pat. No. 4,950,665.

This invention is generally in the area of methods for the treatment ofviral diseases, and more specifically relates to the treatment of AIDSusing thiazine dyes.

Methylene blue, 3,7-Bis(dimethylamino)-phenothiazin-5-ium chloride, C₁₆H₁₈ ClN₃ S, is a dark green or blue thiazine dye which was firstisolated in 1876. It is FDA approved for oral administration and hasbeen reported to be effective as an antiseptic, disinfectant, andantidote for cyanide and nitrate poisoning. For over 50 years it hasbeen known that methylene blue is reduced by mitochondria to leukodyewhich is then auto-oxidized back to methylene blue by oxygen, yieldingH₂ O₂. This is the probable mechanism by which methylene blue, injectedi.v. at a dose of 1 mg/kg body weight, is effective in the treatment ofmethemoglobinemia, a clinical disorder where more than 1% of thehemoglobin in the blood has been oxidized to Fe³⁺. Kelner and Alexanderreported in J. Biol. Chem. 260(28), 15168-15171 (1985), that methyleneblue oxidizes glutathione directly when it is reduced by NADPH, ratherthan via the H₂ O₂.

Methylene blue, in the presence of light, has been reported to damageDNA, probably by destroying or cleaving the DNA at the guanine residues.Simon and Van Vunakis, Arch. Biochem. Biophys. 105, 197-206 (1964),noted that the effect of several photoactive dyes and light is dependenton the concentration of the dye, as well as light wavelength andintensity, and can be correlated with uptake of oxygen and decrease inultraviolet absorbance by guanine derivatives. Kornhauser, et al.,Photochem. Photobiol. 18, 63-69 (1973) attempted to characterize thechanges in guanosine following exposure to methylene blue and lightusing thin layer chromatographic analytical techniques.

Others have also attempted, without success, to analyze the actualmechanism of the effect of methylene blue and light on DNA. Friedmannand Brown, Nucleic Acids Res. 5, 615-622 (1978), showed that methyleneblue and light caused lesions at deoxyguanosines in DNA and thatsubsequent exposure to piperidine caused strand rupture. Theyhypothesized that cyclo-addition occurred at various positions in thepurine ring, rendering the DNA susceptible to base catalyzed cleavagefollowing modification of the other nucleoside bases.

Waskell, et al., reported in Biochim. Biophys, Acta 129, 49-53 (1966),that extensive irradiation of polynucleotides in the presence ofmethylene blue causes extensive destruction of guanosine, leavingribose, guanidine, ribosylurea, and free urea. They postulated that thedestruction of the guanosine residues was the mechanism for a previousobservation by Sastry, et al., Biochim. Biophys. Acta 129, 42 (1966),that, in vitro, methylene blue and irradiation inactivates TobaccoMosaic Virus (TMV) RNA, rendering the virus uninfective. Singer andFraenkel-Conrat, have also reported, in Biochem. 4, 2446-2450 (1966),that another type of dye, thiopyronin (where the ring N is replaced byCH), and proflavin, cause inactivation of TMV RNA in the presence oflight. This is also the probable mechanism for the observation thattopical administration of a 0.1% solution of methylene blue inconjunction with polychromatic light photoinactivates viruses such asherpes simplex, as referenced in American Hospital Formulary Service92:00 Unclassified Therapeutic Agents, page 2176 editor, Gerald K.McEvoy (American Society of Hospital Pharmacists, Inc. 1981 revised1988). Other observations have been made showing in vitro inactivationof viruses using light, methylene blue, and electricity, as reported inJ. Clin. Microbiol., 17(2), 374-376 (1983), by Badylak, et al.,(pseudorabies virus) and Proc. Soc. Exper. Biol. Med. 161,204-209 (1979)by Swartz, et al., (Herpes simplex).

U.S. Pat. No. 4,950,065 entitled "Phototherapy Using Methylene Blue"filed Oct. 28, 1988 by Robert A. Floyd disclosed a method for usingthiazine dyes, especially methylene blue, in combination with light tohydroxylate guanosine or deoxyguanosine at the C8 of the purine ring.The number of guanosines in a nucleic acid strand converted to8-OH-deoxyguanosine (8-OH-dG) or 8-OH-guanosine (8-OH-G) can becontrolled through manipulation of the concentration of methylene blue,light intensity and length of exposure, pH, and buffer strength. Verylittle, if any, other derivatives are formed. As described, the methodcan be used for the selective mutation or modification of either a DNAor a RNA sequence, or the protein expressed therefrom. 8-OH-dG and8-OH-G do not base pair well and are especially susceptible tomisreading.

Hydroxylation of guanine in DNA to produce 8-hydroxydeoxyguanosine(8-OH-dG) has been postulated to be an important factor in mutation andcarcinogenesis by Kasai and Nishimura, Nucleic Acid Res. 12, 2137-2145(1984); Gann. 75, 565-566 and 841-844 (1984); Environ. Health Perspect.67, 111-116 (1986); Kasai, et al., Gann. 75, 1037-1039 (1984);Carcinogenesis 7, 1849-1851 (1986); Aida and Nishimura, Mutation Res.192, 83-89 (1987). Kuchino, et al., Nature (London) 327, 77-79 (1987)used synthetic oligonucleotides containing 8-hydroxydeoxyguanosine in aspecific position as a template for DNA synthesis to show misreading atthe modified base and at adjacent pyrimidine bases. They observed thatspecific base-pairing was completely lacking at the 8-hydroxyguanosineand that incorrect bases were inserted at the adjacent pyrimidine bases.Kasai, et al., reported in Carcinogenesis 8(12), 1959-1961 (1987) thatadministration of a renal carcinogen, potassium bromate, to the ratcaused a significant increase of 8-hydroxydeoxyguanosine in the kidneyDNA, but not in non-target organ DNA.

Chemically, 8-hydroxydeoxyguanosine is made from guanosine by the actionof reagents which generate oxygen radicals, such as ascorbic acid andother reducing agents, metals, polyphenols, and asbestos, and byx-irradiation. Intracellular DNA appears to undergo repair by enzymesfollowing formation of 8-hydroxy-deoxyguanosine. This may be a naturallyoccurring response which has evolved to combat the effects of the manymutagens, tumor promoters, and carcinogens which cause the formation of8-hydroxydeoxyguanosine.

As described in U.S. Ser. No. 264,088, issued as U.S. Pat. No.4,950,665, on Aug. 21, 1990, the selective administration of thiazinedyes can be used in the treatment of viral infections and in cancer.Selective delivery can be achieved using systems such as liposomes fordelivery to macrophages and other phagocytic cells or biodegradablecontrolled release implants. Viruses, bacteria, and cells undergoingrapid DNA synthesis are all inactivated by methylene blue whenirradiated with light. Treatment can be extracorporeal or by lightirradiation of specific tissues using other methods. Methylene blueabsorbs in the red wavelengths, i.e., approximately 670 nm, whichpenetrates tissue much better than other lower wavelengths.

Acquired Immunodeficiency Syndrome (AIDS) is generally accepted at thistime to be a consequence of infection with the retrovirus variouslytermed human T-lymphotropic virus type III (HTLV-III),lymphadenopathy-associated virus (LAV), AIDS associated retrovirus(ARV), or human immunodeficiency virus (HIV-1). There is considerabledifficulty in diagnosing the risk of development of AIDS. AIDS is knownto develop in at least 50% of the individuals infected with humanimmunodeficiency virus (HIV), although this percentage is suspected tobe much higher.

A patient is generally diagnosed as having AIDS when a previouslyhealthy adult with an intact immune system acquires impaired T-cellimmunity. The impaired immunity usually appears over a period ofeighteen months to three years. As a result of this impaired immunity,the patient becomes susceptible to opportunistic infections, varioustypes of cancer such as Kaposi's sarcoma, and other disorders associatedwith reduced functioning of the immune system.

No treatment capable of preventing or curing HIV infection is currentlyavailable, although several compounds have demonstrated antiviralactivity against the virus in vitro, including HPA-23, interferons,ribavirin, phosphonoformate, ansamycin, suramin, imuthiol,penicillamine, rifabutin, AL-721, 3'-azido-3'-deoxythymidine (AZT), andother 2',3'-dideoxynucleosides. AZT is the only drug which has beendemonstrated to prolong life of patients infected with HIV. However, AZTis quite toxic when used for periods of several months and must bediscontinued even in those patients initially tolerant to the drug dueto the drug causing severe anemia. See Yarchoan et al., Lancet, 575-580(1986). Further, AZT-resistant strains of HIV-have now been reported inpatients undergoing treatment with AZT, Larder, B. A., Darby, G.,Richmond, D. D., Science 243, 1731-1734 (1989).

Many inhibitors of cellular processes, such as AZT, limit viralreplication, but are at the same time quite toxic for the host as well.Most of the antiviral drugs that have been discovered so far cannot beprescribed for a prolonged period of time because of their toxicity.Accordingly, it is clear that there is a strong need for new antiviralagents, especially those with low toxicity to normal cells. Moreparticularly, because of the high mortality of AIDS and the lack of aneffective treatment for this disease, there remains a great need fordevelopment of new low toxicity agents for prophylactic use as well aslong term therapy of AIDS patients.

It is therefore an object of the present invention to provide methodsand compositions for treatment or prevention of viral infections.

It is further object of the present invention to provide methods andcompositions for selectively inactivating HIV.

SUMMARY OF THE INVENTION

A method for using thiazine dyes, especially methylene blue, alone or incombination with low levels of light, to selectively inactivate orinhibit intracellular replication of specific viruses, especially humanimmunodeficiency virus.

Examples of useful thiazine dyes are methylene blue, azure A, azure C,toluidine blue O, and thionine. The preferred dye at this time ismethylene blue. Methylene blue is FDA approved for topical, i.v., andoral administration, and has minimal side effects. Selective deliverycan be achieved using systems such as liposomes for delivery tomacrophages and other phagocytic cells or using biodegradable controlledrelease implants. Since methylene blue absorbs in the red wavelengths,i.e., approximately 670 nm, which penetrates tissue much better thanother lower wavelengths, light penetrating the skin to the capillariesat the surface can be used to enhance the activity of the dye.

BRIEF DESCRIPTION OF THE DRAWINGS

FIGS. 1A and 1B are graphs of the effect of methylene blue in the lightand in the dark on the replication of HIV-1 in human PBM cells, %inhibition versus log concentration of methylene blue, μM, FIG. 1A is abar graph; FIG. 1B is a line graph.

DETAILED DESCRIPTION OF THE INVENTION

In U.S. Pat. No. 4,950,665 entitled "Phototherapy Using Methylene Blue"filed Oct. 28, 1988, by Robert A. Floyd, issued as U.S. Pat. No.4,950,665 on Aug. 21, 1990 it was demonstrated that the thiazine dyemethylene blue plus light hydroxylates both guanosine and deoxyguanosineto yield 8-OH-guanosine (8-OH-G) and 8-OH-deoxyguanosine (8-OH-dG),respectively. This treatment can be used to selectively, and in acontrolled manner, to modify the guanine bases in viral DNA and RNA,both in vitro and intracellularly. 8-OH-G is used herein to refer toboth 8-OH-G and 8-OH-dG unless otherwise stated.

The production of 8-OH-G results in mutations in nucleic acid since itdoes not base-pair as well as the unaltered guanosine and because thebases adjacent to the 8-OH-G can be misread during replication,transcription, and translation. Viruses replicate within the host'scells, using enzymes encoded by their own genetic material and the hostcell's "machinery". Replication usually occurs at a high rate, with muchof the transcription and translation being involved in production of newvirus. It has now been discovered that the dyes interfere withtranscription and translation in the dark or in low light levels, aswell as in light. Further, it has been discovered that some types ofviruses are particularly sensitive to the thiazine dyes. Examples ofthese viruses are human immunodeficiency virus (HIV), and, to a lesserdegree, Herpes simplex virus (HSV). Accordingly, the thiazine dyes canbe used to treat infections by these viruses in vivo by interfering withthe transcription and translation involved in replication of the virusin the host cells, at least in part by the mechanism whereby the dyehydroxylates the guanosine or deoxyguanosine to yield 8-OH-guanosine(8-OH-G) or 8-OH-deoxyguanosine (8-OH-dG), respectively. There isminimal effect on the host cell, presumably because of the lower rate ofreplication.

The activity of the dye can be enhanced further by irradiation withlight or by derivatization with compounds such as antisense mRNA. Thethiazine dye can also be provided in combination with other knownantibiotics, anti-inflammatories, antifungals, and antivirals.

Thiazine dyes have been used in a variety of applications, as discussedin the Background of the Invention. However, the method described hereinis based on the selective and controlled use of the compound, not thecomplete destruction of all genetic material, which would be completelyinapplicable to any method wherein the goal is to preserve and minimizetoxic effects to the host cells. Further, the method described hereindoes not require administration of exogenous light, although the resultsmay be enhanced by exposure to light in addition to that normallytransmitted through the skin.

Examples of useful thiazine dyes are methylene blue, azure A, azure B,toluidine blue O, and thionine. Methylene blue is the preferred dye atthis time. These dyes are all commercially available from a number ofdifferent sources.

The dyes can be applied topically or systemically for in vivoapplications. Both methods of administration are approved by the FederalDrug and Food Administration for methylene blue, although methylene blueis not FDA approved at this time for any topical or in vivo applicationfor the treatment of viral infections. Methylene blue is a thiazine dyeoccurring as dark blue-green crystals which is soluble in water andsparingly soluble in alcohol, forming deep blue solutions. Methyleneblue injectable has a pH of 3-4.5. The pK_(a) is between 0 and -1.

Drug Facts and Comparisons, page 1655 (J. B. Lippincott Co., St. Louis,Mo. 1989) reports that methylene blue is useful as a mild genitourinaryantiseptic for cystitis and urethritis, in the treatment of idiopathicand drug-induced methemoglobemia and as an antidote for cyanidepoisoning. Recommended dosages are 55 to 130 mg three times daily,administered orally. Oral absorption is 53% to 97%, averaging 74%,DiSanto and Wagner, J. Pharm. Sci. 61 (7), 1086-1090 (1972).Pharmacopeia states that the recommended dose is 50 to 300 mg by mouth;1 to 4 mg/kg body weight i.v. Side effects include blue urine,occasional nausea, anemia and fever. American Hospital Formulary Service"Drug Information 88" states that the recommended i.v. dosage forchildren is 1 to 2 mg/kg body weight, injected slowly over severalminutes, which can be repeated after an hour. 55 mg tablets areavailable from Kenneth Manne. 65 mg tablets are available from StarPharmaceuticals. Methylene Blue Injection (10 mg/ml) is available fromAmerican Reagent, Harvey, Kissimmee, Pasadena.

Narsapur and Naylor reported in J. Affective Disorders 5, 155-161 (1983)that administration of methylene blue orally, at a dosage of 100 mgb.i.d. or t.i.d., or intravenously, 100 mg infused over 10 min, may beeffective in treating some types of mental disorders in humans,indicating that the dye may cross the blood-brain barrier and thereforehave particular applicability in the treatment of viral infections ofthe brain and central nervous system. Methylene blue was administeredfor periods of one week to 19 months to adult humans, with minimal sideeffects.

The American Hospital Formulary Service "Drug Information 88" reportsthat methylene blue is absorbed well from the GI tract, with about 75%excreted in urine and via the bile, mostly as stabilized colorlessleukomethylene blue. As reported by G. E. Burrows in J. Vet. Pharmacol.Therap. 7, 225-231 (1984), the overall elimination rate constant ofmethylene blue, in sheep, is 0.0076±0.0016 min⁻¹ with minimalmethemoglobin production at doses as high as 50 mg/kg and no hematologicchanges seen up to four weeks after a total dose of 30 mg/kg methyleneblue. The 24 h LD₅₀ for intravenous methylene blue administered as a 3%solution was 42.3 mg/kg with 95% confidence interval limits of 37.3 to47.9 mg/kg, demonstrating that methylene blue can be safely administeredat a dosage of up to at least 15 mg/kg. As reported by Ziv and Heavnerin J. Vet. Pharmacol. Therap. 7,55-59 (1984), methylene blue crosses theblood-milk barrier easily.

The method described herein for the inhibition of HIV infections invivorequires dosages in the range producing a blood concentration ofapproximately 20 to 200 micromolar, or 7.5 to 75 mg/l. The usual bloodvolume for babies is approximately 2.5 l, for adult humans it isapproximately 10 l. Taking into account the 74% oral absorption and 75%excretion of that absorbed over a period of time, and assuming the lowertherapeutic index in darkness than in light, this is approximatelyequivalent to 5.76 mg/kg over an 18 hour period.

The thiazine dyes can also be delivered using techniques known to thoseskilled in the art of drug delivery to target specific cell types or toenhance the activity of the dye. For example, a procedure utilizinginjection of photoactive drugs for cancer treatment is described byEdelson, et al., in New England J. Med. 316, 297-303 (1987). Thiazinedyes can be specifically delivered to macrophages, a site of high HIVconcentration in AIDS patients, using techniques such as liposomedelivery. Liposomes are generally described by Gregoriadis, DrugCarriers in Biology and Medicine Ch. 14, 287-341 (Academic Press, NY,1979). Methods for making light sensitive liposomes are described byPidgeon, et al., in Photochem. Photobiol. 37, 491-494 (1983). Liposomecompositions are commercially available from companies such as theLiposome Company, Inc., Princeton, N.J. Release of compounds fromliposomes ingested by macrophages is described by Storm, et al., inBiochim. Biophys. Acta 965, 136-145 (1988).

Alternatively, the dye can be continuously delivered to a patient overan extended period of time using a controlled release polymeric implant.Polymeric implants are generally manufactured from polymers whichdegrade in vivo over a known period of time. Examples of useful polymersinclude polyanhydrides, polylactic acid, polyorthoester, and ethylenevinyl acetate. These devices are also commercially available. AlzaCorporation, Palo Alta, Calif., and Nova Pharmaceuticals, Baltimore,Md., both manufacture and distribute biodegradable controlled releasepolymeric devices.

The present invention will be further understood with reference to thefollowing non-limiting examples.

EXAMPLE 1 Selective Formation of 8-hydroxyguanine using methylene blueand light

Methylene blue plus light causes formation of8-hydroxy-2'-deoxyguanosine (8-OHdG). The amount of 8-OHdG formed in DNAby methylene blue plus light increases as a function of time of exposureto white light and as the methylene blue concentration increases. Theprocedure used was as follows.

Methylene blue was added to calf thymus DNA solubilized in sodiumphosphate buffer (0.0935 mg DNA/ml, 0.014M Na Phosphate, pH 6.8) toproduce a final concentration of 0.02 mM. The methylene blue containingDNA solution was then exposed to white light (100 watt incandescentlight 11 cm from a beaker containing the sample being treated) in an icebath. The treated DNA was precipitated with ethanol (400 μl 5M NaCl in 5ml ethanol), collected by centrifugation at 2000 rpm for one hr at 4°C., and the pellet redissolved in 250 μl Bis-Tris EDTA buffer containing10 mM MgCl₂. The redissolved DNA was next placed in a boiling water bathfor three min then cooled rapidly, digested with DNAse I (10 μg) andendonuclease (0.6 units) overnight at 37° C. The DNA was furtherdigested to the free nucleotide level with phosphodiesterase (PDE)(Calbiochem snake venom, 0.04 units) and alkaline phosphatase from calfintestine (Calbiochem, 1 unit) overnight at pH 8.0 and 37° C. The pH ofthe solution was lowered to 4.0 with acetic acid and the volume wasadjusted to 250 μl. The sample was filtered and run on HPLC to measurethe concentration of 8-hydroxyguanosine. Methylene blue plus lightclearly causes hydroxylation of guanine as shown in Table 1.

The nucleosides of adenine, cytosine, thymine, and guanine(approximately 200 nmoles each) were also subjected to methylene blue(20 μl into 2.0 mls final total volume) plus light treatment (100 wattincandescent light 11 cm over solution for 15 min). Only deoxyguanosinereacted to form the hydroxylated derivative, based on HPLC analysis.

Similar results are obtained when RNA is substituted for the DNA.

                  TABLE 1                                                         ______________________________________                                        Quantitation of 8-hydroxyguanosine in DNA                                     exposed to methylene blue and light.                                          Experimental     Ratio of 8-OHdG                                              Condition        to 10.sup.5 dG                                               ______________________________________                                        DNA + Light      6.8                                                          DNA + MB         24.2                                                         DNA + Light + MB 484.2                                                        ______________________________________                                         Data represent the average of 5 replicate samples                        

EXAMPLE 2 Inactivation of RNA virus R17 using methylene blue and light

Exposure of the RNA virus R17 to 0.02 μM methylene blue plus light for15 min (100 watt incandescent bulb at 11 cm) causes inactivation of R17,as assessed by its ability to form plaques on a bacterial lawn. Theinfectivity of the virus is inactivated 50% by a 45 seconds exposure tolight in the presence of 0.05 μM methylene blue.

Add 20 μl φ (R17 phage) stock to 2.0 ml dilution buffer and dispense 0.3ml into each of tubes A-F. Next add methylene blue to giveconcentrations shown in Table 2 below. Pipet 270 μl of samples to belight treated into the #1 wells of a 96 well microtiter plate,corresponding to its letter label A-H. The samples in the microtiterplate were exposed to light through water (1/4 cm) deep in a petri dishfor 5 minutes. 270 μl of each sample not to be light treated was thenadded to wells in the plate.

After treatment, viable R17 phage were titered by making serial10×dilutions in the microtiter plate, adding 0.1 ml of selected dilutionto 0.2 ml of log phase E. coil strain XL-1-Blue cells (approximately 10⁷cells/ml), obtained from Stratagene, La Joya, Calif., and plating out0.1 ml.

                  TABLE 2                                                         ______________________________________                                        Effect of methylene blue and light on the                                     infectivity of RNA virus R17.                                                 MB conc.                                                                      (uM)           Light 5' pfu/ml                                                ______________________________________                                        0              no       7.20 × 10.sup.11                                0.02           yes      5.04 × 10.sup.10                                0.04           yes      1.11 × 10.sup.10                                0.2            yes      3.25 × 10.sup.5                                 2.0            no       4.80 × 10.sup.10                                2.0            yes      3.00 × 10.sup.3                                 20.0           no       3.00 × 10.sup.9                                 20.0           yes      4.20 × 10.sup.4                                 ______________________________________                                    

The methylene blue did not inhibit growth of the bacterial lawn at theconcentrations used in the R17 inactivation and titering.

EXAMPLE 3 Effect of duration of light exposure on the inactivation ofR17 virus

R17 virus samples were also tested to determine the time of exposure oflight required to inactivate the virus. The procedure was similar tothat used for Example 2 as outlined above except that the concentrationof MB was held constant at 0.05 uM while the time of exposure wasvaried. The length of exposure was varied as follows:

Add 0.3 ml to well H2, treat with light 0-5 min. Add 0.3 ml to well G2,treat with light 5 min-7.5 min. Add 0.3 ml to well F2, treat with light7.5 min-9.0 min. Add 0.3 ml to well E2, treat with light 9.0 min-9.5min. Add 0.3 ml to well D2, treat with light 9.5 min-9.9 min. Add 0.3 mlto well C2, treat with light 9.9 min-10.0 min. Add 0.3 ml to well B, nolight. Add 0.3 ml to well A, no light. The results are shown in Table 3.

                  TABLE 3                                                         ______________________________________                                        Effect of time of exposure to light on                                        infectivity of R17 virus treated with MB.                                     Time (Min.)   PFU                                                             ______________________________________                                        none          2.76 × 10.sup.9 /ml                                       0.1'          3.3 × 10.sup.9 /ml                                        0.5'          5.0 × 10.sup.9 /ml                                        1.0'          1.0 × 10.sup.9 /ml                                        2.5'          9.1 × 10.sup.7 /ml                                        5.0'          6.93 × 10.sup.7 /ml                                       10.0'         4.35 × 10.sup.6 /ml                                       ______________________________________                                    

These results demonstrate that methylene blue and light inactivated R17under these conditions at a rate of t.sub. 1/2 =0.8-1.0 minutes.

EXAMPLE 4 Anti-vital Effect of methylene blue on human immunodeficiencyvirus (HIV), Compared with AZT

Methylene blue was prepared as described above. AZT was synthesized andpurified by a modification of the method of Lin and Prusoff (Lin, T. -S,and W. H. Prusoff, J. Med. Chem. 21, 109-112 (1978). Acyclovir (ACV) wasobtained from the Burroughs-Wellcome Co.

HIV Antiviral Studies.

Cells:

Human peripheral blood mononuclear cells (PBMC) from healthy HIV-1seronegative and hepatitis B virus seronegative donors were isolated byFicoll-Hypaque discontinuous gradient centrifugation at 1,000×g for 30minutes, washed twice in phosphate-buffered saline (pH 7.2; PBS), andpelleted at 300×g for 10 minutes. Before infection, the cells werestimulated by phytohemagglutinin (PHA) at a concentration of 16.7 μg/ml,and 4 mM sodium bicarbonate buffer.

Retrovirus:

HIV-1 (strain LAV-1) was obtained from Dr. P. Feorino (Centers forDisease Control, Atlanta, Ga.). The virus was propagated in human PBMCusing RPMI 1640 medium, as described previously by McDougal, et al.,("Immunoassay for the detection and quantitation of infectious humanretrovirus, lymphadenopathy-associated virus (LAV)," J. Immun. Meth. 76,171-183, 1985) without PHA or fungizone and supplemented with 7% (v/v)interleukin-2 (Advanced Biotechnologies, Silver Spring, Md.), 7 μg/mlDEAE-dextran (Pharmacia, Uppsala, Sweden), and 370 U/ml anti-humanleukocyte (alpha) interferon (ICN, Lisle, Ill.). Virus obtained fromcell-free culture supernatant was titrated and stored in aliquots at-70° C. until use.

Inhibition of virus Replication in Human PBMC:

Uninfected PHA-stimulated human PBMC were uniformly distributed among 25cm² flasks to give a 5 ml suspension containing about 2×10⁶ cells/mi.Suitable dilutions of virus were added to infect the cultures. The meanreverse transcriptase (RT) activity of the inocula was 50,000 dpm/mlcorresponding to about 100 TCID₅₀, as determined by Groopman, et al.,(1987), "Characterization of Serum Neutralization Response to the HumanImmunodeficiency Virus (HIV)," AIDS Res. Human Retro. 3, 71-85. Thedrugs, at twice their final concentrations in 5 ml of RPMI 1640 medium(supplemented as described above), were added to the cultures.Uninfected and untreated PBMC at equivalent cell densities were grown inparallel as controls. The cultures were maintained in a humidified 5%CO₂ -95% air incubator at 37° C. for six days after infection at whichpoint all cultures were sampled for supernatant RT activity. Previousstudies had indicated that maximum RT levels were obtained at that time.

RT Activity Assay:

A six milliliter aliquot of supernatant from each culture was clarifiedof cells at 300×g for 10 minutes. Virus particles were pelleted from 5ml samples at 40,000 rpm for 30 minutes using a Beckman 70.1 Ti rotorand suspended in 200 μl of virus disrupting buffer (50 mM Tris-HCl, pH7.8, 800 mM NaCl, 20% glycerol, 0.5 mM phenylmethyl sulfonyl fluoride,and 0.5% Triton X-100).

The RT assay was performed in 96-well microtiter plates, as described bySpira, et al. (1987), "Micromethod for Assaying the ReverseTranscriptase of LAV-HTLV-III/Lymphadenopathy-Associated Virus, " J.Clin. Microbiol. 25, 97-99. The reaction mixture, which contained 50 mMTris-HCl, pH 7.8, 9 mM MgCl₂, 5 mM dithiothreitol, 4.7 μg/ml (rA)_(n)·(dT)₁₂₋₁₈ 140 μM dATP, and 0.22 μM [³ H]TTP (specific activity 78.0Ci/mmol, equivalent to 17,300 cpm/pmol; NEN Research Products, Boston,Mass.), was added to each well. The sample (20 μl) was added to thereaction mixture which was then incubated at 37° C. for 2 hours. Thereaction was terminated by the addition of 100 μl 10% trichloroaceticacid (TCA) containing 0.45 mM sodium pyrophosphate. The acid-insolublenucleic acids which precipitated were collected on glass filters using aSkatron semi-automatic harvester. The filters were washed with 5% TCAand 70% ethanol, dried, and placed in scintillation vials. Four ml ofscintillation fluid (Econofluor, NEN Research Products, Boston, Mass.)were added and the amount of radioactivity in each sample was determinedusing a Packard Tri-Carb liquid scintillation analyzer (model 2,000 CA).The results were expressed in dpm/ml of original clarified supernatant.The procedures for the anti-HIV-1 assays in PBMC described above havebeen published recently (see Schinazi, et al., Antimicrob. AgentsChemother. 32, 1784-1789, December 1988).

HSV Preparation:

Confluent HEp-2 cells, in a roller bottle (Falcon, 850 cm²), wereinfected at an input multiplicity of 0.01 PFU per cell (to minimizeproduction of defective viruses). During the two-hour absorption period,the cells were exposed to the virus inoculum diluted in absorptionbuffer (10 ml PBS containing 1% newborn calf serum and 0.1% glucose).The virus inoculum was then removed and replaced with Hanks' MinimumEssential Medium (MEM) containing 2% inactivated newborn calf serum,penicillin (100 U/ml) and streptomycin (100 μg/ml) (P and S), sodiumbicarbonate (2 g/L) and HEPES (25 mM) (maintenance medium). The infectedcells were incubated at 37° C. for 3 to 4 days, until the cells could beeasily shaken from the plastic surface of the culture bottles. The cellswere then collected by centrifugation, suspended in a small volume ofspent culture fluid (5 ml per 2×10⁸ cells) and sonicated 3 times for onemin on ice. The disrupted cells were centrifuged (2,000 g for 15 min at4° C. and the supernatant was diluted with an equal volume of sterileskimmed milk (as stabilizer) and aliquots were frozen at -70° C.

HSV Plaque Reduction Assay:

Near confluent Vero (African Green Monkey) cells in 6-well plates wereinfected with 100 μl virus diluted in absorption buffer to give 100-200plaques per well. The plates were then incubated at 37° C. for 1 hourwith intermittent rocking every 15 minutes. The inoculum was aspiratedand the compounds at different concentrations (dissolved in maintenancemedium) were added to replicate wells. For these assays, 0.1% pooledhuman gamma globulin was included in the media. The plates were placedin 5% CO₂ -95% air incubator and the plaques were allowed to develop for48 h prior to fixation (buffered 10% Formalin acetate), staining (0.5%crystal violet in 20% EtOH/H₂ O) and enumeration. The degree ofinhibition (per cent plaques of control) was calculated by counting themean plaque counts for the different drug dilutions. The antiviralpotency of the drugs was determined by estimating the ED₅₀, the drugconcentration necessary to reduce the number of plaques by 50% relativeto the virus control cultures. For routine antiviral drug screening, weused the F strain of HSV-1 and the G strain of HSV-2 (Ejercito, et al.,"Characterization of Herpes Simplex Virus Strains Differing in TheirEffect on Social Behavior of Infected Cells," J. Gen. virol, 2, 357-364(1968)). For HSV plaquing, Vero cells (rather than a human cell linesuch as fibroblasts) were used since these cells do not induceinterferon. Acyclovir (ACV) was used as a positive control for thestudies on HSV.

Cytotoxicity studies.

Toxicity in Veto (African Green Monkey) Cells:

Vero cells in growth medium (2.5 ml) were added to 25 cm² flasks(Falcon) in duplicate at a concentration equivalent to one-tenth of cellconfluency for each compound under test. After incubation at 37° C. in5% CO₂ -95% air for 24 hr, the test compound (2×final concentration),dissolved in 2.5 ml of the growth medium was added, and two flasks wereharvested immediately by decanting the medium, washing once with 3 ml ofPBS, and then incubating at 37° C. for 5 min with 3 ml of trypsin/EDTA(0.125%/0.02%). The cells dislodged from the flask were generally inclumps and were dispersed by repeated forceful pipetting of thesuspension against the surface of the flask. To 1 ml of thewell-dispersed cell suspension, 0.2 ml of trypan blue solution wasadded, and the number of cells were counted using a hemacytomer. Eachday for the next 3 days, two of the remaining flasks were harvested inthe manner just described for determination of cell number. Only data onday three are presented. This method has previously been described bySchinazi, et al., "Effect of Combination of Acyclovir, and Vidarabine orits 5'-monophosphate on Herpes Simplex Viruses in Cell Culture and inMice," Antimicrob. Agents Chemother. 22, 499-507 (1982)).

PBM and CEM Cells Proliferation Assay:

The drugs were evaluated for their potential toxic effects on uninfectedPHA-stimulated human PBM cells and also in CEM cells. The cells werecultured with and without drug for 6 days at which time aliquots werecounted for cell viability as described above.

Enzyme Assays:

The preparation of the enzyme and the assay conditions used, wererecently described by Schinazi, et al., Antimicrob. Agents Chemother.33, 115-117 (1989). HIV-1 RT and cellular DNA polymerase alpha wereisolated from infected and uninfected PHA-stimulated human PBM cellsaccording to traditional methods (Eriksson, B., et al., Antimicrob.Agents Chemother. 31, 600-604, (1987); Furman, et al., Proc. Natl. Acad.Sci, USA 83, 8333-8337, (1986); Abrell, et al., J. Virol. 12, 431-439,(1973).

    ______________________________________                                        Assays. Reaction mixtures (100 μl) contained:                                            RT           DNA pol. alpha                                     ______________________________________                                        Tris-HCl, pH 8.0                                                                              100    mM      100   mM                                       KCl             50     mM      --                                             MgCl.sub.2      2      mM      6     mM                                       DTT             5      mM      5     mM                                       BSA             400    μg/ml                                                                              400   μg/ml                                 [.sup.3 H]dTTP, 1      μM   1     μM                                    (Sp.act. 82.3 Ci/mmol)                                                        dATP, dCTP, dGTP                                                                              --             100   μM                                    Poly (rA)-oligo (dT).sub.12-18                                                                3      μg/ml                                                                              --                                             Activated DNA   --             200   μg/ml                                 ______________________________________                                    

The reactions were started by the addition of 10 μl of purified enzyme,incubated at 37° C. for the indicated periods of time and processed asdescribed in Eriksson, et al., Antimicrob. Agents Chemother. 31, 600-604(1987).

Median-Effect Method:

EC₅₀ and IC₅₀ values were obtained by analysis of the data using themedian-effect equation (Chou, T., et al., "Quantitative Analysis ofDose-Effect Relationships: The Combined Effects of Multiple Drugs orEnzyme Inhibitors," Adv. Enz. Regul. 22, 27-55 (1984).

Results.

The effect of the thiazine dye methylene blue on cellular growth andproliferation was tested using PBM, Vero and CEM cell cultures. Thecytotoxicity of MB, both in the presence and absence of light, wascompared with that of AZT. The antiviral effect of MB was also examinedusing HIV-1, HSV-1 and HSV-2. The combined results of the cytotoxic andantiviral studies are shown in Table 4.

                                      TABLE 4                                     __________________________________________________________________________    Summary of antiviral and cytotoxicity studies on HIV                          and HSV Comparing Thiazine dyes: methylene blue,                              toluidine blue O, azure A, azure B, and thionin, AZT,                         and ACV.                                                                      __________________________________________________________________________    Antiviral Effect:     Cytotoxicity:                                           (EC.sub.50 μM)     (IC.sub.50 μM)                                       Treatment                                                                           HIV-1 HSV-1                                                                              HSV-2                                                                              PBMC.sup.a                                                                         Vero.sup.b                                                                          CEM.sup.c                                    __________________________________________________________________________    AZT   0.00022                                                                             >100 >100 74.6 39.5  56.1                                         MB    0.028 ≧10                                                                         ≧10                                                                         6.1  0.14  0.12                                         light                                                                         MB    0.14  ≧10                                                                         ≧10                                                                         >1.0 0.66  2.22                                         dark                                                                          ACV   >100  0.022                                                                              0.51 >100 1,700 >100                                         light                                                                         ACV         0.013                                                                              0.16 --   --    --                                           dark                                                                          __________________________________________________________________________           Anti-HIV-1                                                                    Effect:                                                                       (EC.sub.50 μM)                                                             Light                Dark                                              __________________________________________________________________________    Toluidine                                                                            0.27                 0.25                                              Blue O                                                                        Azure A                                                                              0.49                 0.39                                              Azure B                                                                              1.8                  5.3                                               Thionine                                                                             1.2                  2.8                                               __________________________________________________________________________     a. PBMC were counted after drug exposure for 6 days by the trypan blue        exclusion method. Untreated cultures had 2.06 × 10.sup.5 cells/ml.      b. Vero cells were counted after drug exposure for 4 days. Untreated          cultures had 3.32 × 10.sup.5 cells/ml.                                  c. CEM cells were counted after drug exposure for 4 days. Untreated           cultures had 1.49 × 10.sup.5 cells/ml.                             

EXAMPLE 5 Measurement of P24 to quantitate inactivation of HIV bymethylene blue.

The efficacy of methylene blue as an antiviral agent was furtherdemonstrated using an independent assay method. While the previousexample measured RT activity, the present example utilizes a directquantitation of a viral protein (P24) as an indicator of antiviraleffectiveness.

Peripheral blood mononuclear (PBM) cells were infected with HIV andtreated with methylene blue as described above. The EC₅₀ levels werecalculated from enzyme immuno-assay (EIA) measurements of viral coatprotein P24. The P24-specific EIA kit was purchased from Abbott Labs. Asin Example 4, the effectiveness of methylene blue was compared with AZT,the results are shown in Table 5.

                  TABLE 5                                                         ______________________________________                                        Effect of MB against HIV (LAV-I) in human                                     PBMC Determination of P24 Levels by                                           EIA (Abbott Ag Kit)                                                                   Conc.              % Inhibition                                                                           EC.sub.50                                 Treatment                                                                             μM   ng P24/ml  (corrected)                                                                            μM                                     ______________________________________                                        Uninfected      -0.1                                                          control                                                                       Infected        336.0                                                         control         370.5                                                                         370.5                                                                 mean    359.0      0.0                                                AZT     0.0001  360.7      -0.5                                                       0.001   266.2      25.8                                                       0.01    36.1       89.9     0.0064 μM                                      0.1     5.2        98.5                                               M.B. Light                                                                            0.001   370.5      -3.2                                                       0.01    370.5      -3.2                                                       .1      302.2      15.8                                                       1       5.1        98.6     0.024                                             10      2.2        99.4                                               M.B. Dark                                                                             0.001   356.0      0.8                                                        0.01    270.2      24.7                                                       .1      287.2      20.0                                                       1       84.1       76.6     0.33                                              10      5.4        98.5                                               ______________________________________                                    

EXAMPLE 6 The effect of methylene blue on isolated DNA polymerase alphaand HIV reverse transcriptase.

The effect of methylene blue on two DNA polymerizing enzymes, HIVreverse transcriptase (RT) and calf thymus DNA polymerase alpha (CT αpol), was also studied. The results clearly show that MB is effective atinhibiting reverse transcriptase mediated DNA polymerization. In orderto inhibit DNA polymerase alpha directed DNA synthesis, ten fold more MBis required, as shown in Table 6.

                  TABLE 6                                                         ______________________________________                                        Effect of MB on HIV-1 reverse transcriptase                                   (RT) and calf thymus DNA polymerase in the                                    presence and absence of light.                                                               IC.sub.50 (μM)                                              Enzyme           Light   Dark                                                 ______________________________________                                        HIV-1 RT         9.2     10.0                                                 CT α pol.  125     105.1                                                ______________________________________                                    

Modifications and variations of the method to selectively, and in acontrolled manner, inhibit specific viruses such as HIV, and use thereofin the treatment of viral infections will be obvious to those skilled inthe art from the foregoing detailed description. Such modifications andvariations are intended to come within the scope of the appended claims.

We claim:
 1. A method for treating a biological fluid, obtained from ahuman for administration to a human patient in need thereof, in order toinactivate human immunodeficiency virus in the biological fluidcomprising the steps of:adding to the biological fluid a thiazine dyeselected from the group consisting of methylene blue, toluidine blue O,azure A, azure B, azure C and combinations thereof, in a concentrationwhich is effective to inactivate the human immunodeficiency virus in thebiological fluid upon exposure to light, and exposing the biologicalfluid to light of a wavelength and a period of time effective toactivate the dye which induces the formation of 8-hydroxyguanosine insaid virus.
 2. The method of claim 1 wherein the dye is methylene blue.3. The method of claim 1 wherein the biological fluid is blood or bloodcomponents.
 4. The method of claim 3 wherein the blood componentscomprise peripheral blood mononuclear cells.
 5. The method of claim 1wherein the light is administered at an energy which is approximatelyequivalent to the redwavelenght light received by a sample exposed to a100 Watt incandescent light bulb eleven centimeters from the sample forat least 45 seconds.
 6. The method of claim 1 where the light isequivalent to at least 15 minutes exposure to a 100 Watt incandescentlight eleven centimeters from the sample.
 7. The method of claim 1wherein the dye is in a concentration of between 0.1-10 μM in thebiological fluid.
 8. The method of claim 1 wherein the light comprisesred wavelengths.
 9. The method of claim 8 wherein the light is at awavelength of about 670 nm.